Uric acid is a product of purine metabolism in birds, reptiles, and primates, including humans. Uric acid is produced in the liver by oxidation of xanthine and hypoxanthine. Xanthine is an intermediate in the catabolism of guanine nucleotides while hypoxanthine is produced during the breakdown of adenine nucleotides. In most mammals, uric acid is further oxidized by the enzyme urate oxidase to allantoin. Allantoin, because of its lost pyrimidine ring, shows a more than 20 times greater water solubility than uric acid.
Urate oxidase, also called uricase, is an enzyme of the purine degradation pathway. Uricase catalyzes the conversion of uric acid+O2 into allantoin+CO2.
Humans lack uricase and do not produce allantonin. This is the result of a mutation that introduces a premature termination codon in the coding sequence of the human uricase gene (Wu et al., J. Mol. Evol. 34:78-84, 1992; Wu et al., Proc Natl Acad Sci USA 91:742-6, 1994, each of which are incorporated by reference). As a consequence of this mutation, purine catabolism in humans terminates with the production of uric acid which is relatively insoluble (the solubility index in distilled water is 13.2 mg/dl) making humans susceptible to a pathological condition known as hyperuricemia. Renal handling of uric acid is complex and requires glomerular filtration, reabsorption of filtered urate, tubular secretion, and finally postsecretory reabsorption.
Hyperuricemia is defined as occurring when the serum level of uric acid is above 8 mg/dl. Hyperuricemia can result in the formation of uric acid crystals in the serum, which can precipitate in joints, skin and kidneys. This can result in inflammation of the joints (gout), renal failure, metabolic acidosis, and hyperkalemia. Overproduction of uric acid can have a variety of origins, including congenital metabolic defects, Lesch-Nyhan syndrome, excess ingestion of purine or proteins, and treatments with uricosuric drugs (Kelley, W. N. and Wortman, R. L. Textbook of Rheumatology, 5th edition, pp 1313-1351, 1997; which is incorporated by reference). Hyperuricemia is also found in patients that have had heart or kidney transplants and are being treated with immunosupressive agents. Hyperuricemia can lead to the loss of kidney function in these patients and can produce significant morbidity and mortality.
Hyperuricemia is also found in patients with malignant diseases. Chemotherapy and radiation therapy of cancer patients can induce a life-threatening condition known as tumor lysis syndrome (Kalemkerian, G. P., Darwish, B., and Varterasian, M. L. Am J. Med. 103, 363-367, 1997; Lorigan, P. C., Woodings, P. L., Morgenstern, G. R., and Scarffe, J. H. Ann Oncol. 7, 631-636, 1996; Hande, K. R., and Garrow, G. C. Am. J. Med. 94, 133-139, 1993). Hematologic malignancies, such as leukemias and lymphomas, are responsible for most cases of tumor lysis syndrome. Tumor lysis syndrome is characterized by the rapid development of hyperuricemia, hyperkalemia, hyperphosphatemia, and acute renal failure. Acute renal failure is the result of the intrarenal precipitation of uric acid. Tumor lysis syndrome is often triggered by cell death induced by chemotherapy or radiotherapy, resulting in the release of intracellular substances. However, occasionally cancer patients with a heavy tumor burden may exhibit hyperuricemia and other features of tumor lysis syndrome even in the absence of radiotherapy or chemotherapy because of the high turnover of malignant cells with subsequent catabolism of released purines into uric acid.
Therapy for the prevention and treatment of the acute renal failure associated with tumor lysis syndrome is a considerable challenge and is currently unsatisfactory for a number of reasons. Methods of treating hyperuricemia include hydration, urinary alkalinization, osmotic diuresis and allopurinol therapy. However, the difficulty in the treatment of hyperuricemia lies in the potential for aggravating other consequences of tumor lysis syndrome. For example, alkalinization of urine to increase uric acid solubility facilitates precipitation of calcium phosphate. Allopurinol (4-hydroxypurinol), an analogue of xanthine, has long been the standard treatment for hyperuricemia and also for the prevention of tumor lysis syndrome. Allopurinol is converted to oxypurinol, which then binds to and inhibits xanthine oxidase, the enzyme that catalyzes the conversion of hypoxanthine and xanthine to uric acid. As a result, uric acid production is inhibited, and xanthine and hypoxanthine concentrations increase. However, allopurinol does not remove uric acid that is already present and deposited intrarenally as crystals. As a result, it is often several days (sometimes 10-14 days) from the initial treatment with allopurinol before a significant decrease in uric acid in serum can be observed. While the solubilities of xanthine and hypoxanthine are only slightly greater than that of uric acid, the purines are catabolized to xanthine, hypoxanthine, and uric acid during allopurinol treatment, rather than mostly to uric acid. The result is that the urinary concentration of uric acid decreases below its solubility and further formation of uric acid crytstals is prevented. However, during excessive catabolism of purines, allopurinol therapy may lead to intrarenal precipitation of hypoxanthine and xanthine, with further aggravation of acute renal failure. This situation has been well documented in clinical practice. Furthermore, allopurinol therapy is also associated with significant toxicity and can cause death. Allopurinol can produce severe toxic effects, including cutaneous hypersensitivity reactions, leukopenia, and hepatomegaly. This drug has been also implicated in the induction of tubulointerstitial nephritis. In addition, allopurinol may cause adverse drug interactions, as has been shown for 6-mercaptopurine and adenine arabinoside, drugs often used to treat lymphoproliferative disease and leukemia (Lauter, C B, Bailey, E J, Lerner, AM. J Infect. Dis 1976; 134, 75-79). Finally, although allopurinol can block the formation of uric acid, it does little to solubilize the uric acid which is already present. All of these adverse effects sometimes make allopurinol ineffective for the treatment of acute hyperuricemia in tumor patients. Thus allopurinol is not an ideal drug for the treatment of hyperuricemia.
Dialysis and continuous ateriovenous hemodialysis are additional methods that are used to remove uric acid in patients with hyperuricemia. However, these treatment methods are problematic in patients with malignancies because of the risk for severe bleeding caused by thrombocytopenia or the need for anticoagulation. Furthermore, because of the invasive nature of these treatments, there is an increased risk for fatal infections in patients who are in many instances immunocompromised due to their illness or therapy they have received.
Uricase has been shown to be an effective treatment for hyperuricemia and tumor lysis syndrome (London, M., and Hudson, P. B. Science, 125, 937-938, 1957; Oberling, F. and Lang, J. M. Nouvelle Presse Med 3, 2026, 1974; Robert, A., Corberand, J. and Regnier, C. Rev. Med. Toulouse 12, 1093-1100, 1976; Masera, G., et. al., J. Pediatrics 100, 152-155, 1982; Jankovic, M., et. al. Am. J. Pediatr. Hematol. Onocol. 7, 202-204, 1985; Masera, G. and Jankovic, M. Ann. Oncol. 8, 407, 1996; Jones, D. P, Mahmoud, H. and Chesney, R. W. Pediatr. Nephrol. 9, 206-212, 1995). Treatment with uricase converts uric acid into the highly soluble allantoin. Furthermore, uricase, if adequately filtered into the urine, may even dissolve already precipitated uric acid crystals and improve renal function.
Uricase has a number of advantages in the treatment of hyperuricemia and nephrolithiasis including the speed of the hypouricemic effect (reduction of hyperuricemia of the order of 50% in less than 24 h) and better protection of the kidney against lithiasis compared with other drugs such as allopurinol.
Uricase is only available in a few countries, currently limiting the use of this therapy. Uricase extracted from Aspergillus flavus through a complex manufacturing process, has been commercially available from Sanofi (Clin-Midy, Paris, France) under the trade name Uricozyme in France since 1975 and in Italy since the early 1980s. This drug has been extensively studied in Europe and has been shown to be very effective in rapidly lowering levels of uric acid in patients within minutes of administration (Oberling, F. and Lang, J. M. Nouvelle Presse Med 3, 2026, 1974; Robert, A., Corberand, J. and Regnier, C. Rev. Med. Toulouse 12, 1093-1100, 1976). This treatment is used very often in patients to prevent tumor lysis syndrome. In one study involving over 400 patients, uricase treatment effectively eliminated tumor lysis syndrome (Masera, G. and Jankovic, M. Ann. Oncol. 8, 407, 1996). In the United States, Uricozyme has been extensively tested by a group of investigators at St. Jude Childrens Hospital and the University of Tennessee (Pui, C.-H., et al. Leukemia, 11, 1813-1816, 1997). These investigators treated 126 children in a 3 year period and found uricase treatment to be much more rapid and effective in reducing serum uric acid levels than allopurinol. Furthermore, none of the uricase treated individuals developed tumor lysis syndrome or required dialysis.
Although oncologists are well aware of the potential advantages of uricase, this therapy has not been widely recognized in the renal community. An obstacle for a more general application of uricase in hyperuricemia may be the complicated manufacturing process of the enzyme involving fermentation, extraction, and purification, which clearly limits its commercial availability, as well as the standardization of enzyme activity. The uricase currently used as a drug is obtained by culturing Aspergillus flavus and isolating the enzyme from the culture medium by extraction, followed by several purification steps. While it is possible to obtain highly purified uricase, this method has disadvantages. Aspergillus flavus is not easy to work with because of its physiology and genetics (WOLOSHUK et al. Applied Environ. Microbiol., 55, 86-90, 1989), making it difficult to obtain strains that can produce substantial amounts of the enzyme. Aspergillus flavus can also produce aflatoxins, which can be difficult to remove during the purification process. The purified uricase must be checked to ensure that it is free from these toxins. In addition, because of the foreign nature of the Aspergillus enzyme, therapy is many times limited to a single dose due to the risk of hypersensitivity reactions.
Although uricase has been tested in the United States, it is not an approved therapy because of the of a high incidence of allergic reactions to this foreign protein. The clinical use of uricase is also compromised by its short circulating half-life. (See, Park et al., Anticancer Res., 1:373-6 (1981).
Hypersensitivity to uricase and the short circulating half-life of uricase can be overcome by the covalent attachment of polyethylene glycol (PEG) (Chen, R. H.-L., et. al. Biochim. Biophys. Acta 660, 293-298, 1981; Nishimara, H., Matsushima, A. and Inada, Y. Enzyme, 26, 49-53, 1981; Savoca, K. V., Davis, F. F. and Palczuk, N. C. Int. Archs. Allegy Appl. Immun. 75, 58-67, 1984; Tsuji, J.-I., et. al. Int. J. Immunopharmac. 7, 725-730, 1985). The attachment of PEG to proteins has been shown to greatly reduce the antigenicity of foreign proteins. For example, formulating a heterologous protein with polyethylene glycol (PEG) to reduce the antigenicity has been proven with the approval of Oncaspar (E. coli Asparaginase). Previous investigators have attached PEG (Molecular Weight 5000) to uricase and successfully treated a small number of patients (Davis, S., Park, Y. K., Abuchowski, A. and Davis, F. F. Lancet 2, 281-283, 1981; Chua, C. C., et. al. Ann. Intern. Med. 109, 114-117, 1988). Davis et. al. (1981) treated patients with a single dose of PEG-uricase (uricase isolated from Candida utilis) (120 IU/m2 surface area, intravenously). Serum uric acid fell to undetectable levels within 60 minutes after injection, and remained undetectable for at least 32 hours. The serum half-life of PEG-5,000 uricase was 6 hours. The half-life of native (unpegylated) uricase was noted to be less than 4 hours in other studies. Precipitating antibodies to PEG-uricase or native uricase were not detected in any patient. Davis et al. noted that PEG-5,000 uricase offered a potentially major therapeutic advantage over native uricase in the treatment of hyperuricemic diseases. Chua, et al. (1988) treated a patient with Non-Hodgkin lymphoma with uricase purified from Arthrobacter protoformiae modifed with PEG-5,000. The patient was treated by intramuscular injection on four separate days. Serum uric acid level fell sharply after each dose. No antibodies to either PEG-5,000 uricase or native uricase were detected through the 26th day post dosing. Chua et al. concluded that PEG-5000 uricase may be useful for treating hyperuricemia in the setting of advanced hematologic malignancies.
There is a need for the more efficient production and formulation of uricase whereby the disadvantages associated with the use of uricase in the treatment of patients with high uric acid levels as discussed above can be overcome. The present invention is directed to these, as well as other, important ends.